Aminocarboxylate ligands having substituted aromatic amide moieties

ABSTRACT

Novel metal-chelate complexes comprising aminocarboxylate ligands including substituted aromatic amide moieties, such as those having the formula 
                         
wherein R 13 , A 1 , R 1  and R 2  are as defined herein, are disclosed.

This application is a continuation of U.S. application Ser. No.10/042,721 filed May 29, 2002, now U.S. Pat. No. 6,875,864, which is acontinuation of U.S. application Ser. No. 08/471,556, filed Jun. 6,1995, now abandoned, which is a divisional of U.S. application Ser. No.08/010,909, filed Jan. 29, 1993, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 07/738,998, filed Aug.1, 1991, now abandoned. All of these applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Metal-chelating ligands are useful in diagnostic medicine as contrastagents. X-ray imaging, radionuclide imaging, ultrasound imaging andmagnetic resonance imaging can each be enhanced by the use of a metalatom bound to a chelating ligand. For example, a chelating ligand canbecome a radiopharmaceutical when it is prepared as a chelate complexwith ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ¹⁴⁰La, ¹⁶⁹Yb, ⁶⁸Ga, ⁹⁰Y, ¹⁸⁸Re, ¹⁵³Sm orother radioactive metal ions. When a chelating ligand is complexed withthe stable isotopes of the lanthanides, tantalum, bismuth or otherelements with molecular weight higher than iodine, the resulting complexabsorbs x-rays sufficiently to act as an x-ray contrast agent. In somecases, the agents that are useful in x-ray imaging absorb, reflect orscatter ultrasound radiation sufficiently to be used as an ultrasoundagent. If a chelating ligand is complexed with a paramagnetic metal atomthat has a symmetric electronic ground state (e.g., Gd⁺³, and octahedralMn⁺², Fe⁺³, Cr⁺³) the resulting complex will be useful as a spinrelaxation catalyst that is used in magnetic resonance imaging (alsoknown as NMR imaging) as a contrast agent. If a chelating agent iscomplexed with a paramagnetic metal atom that has an unsymmetricalelectronic ground state (e.g., dysprosium(III), holmium(III) anderbium(III), the resulting complex will be useful as a chemical shiftagent in magnetic resonance imaging or in magnetic resonance in vivospectroscopy. In addition, any paramagnetic metal ion complex may beused as a contrast agent by virtue of its magnetic susceptibility asdisclosed by villringer et al. (Magnetic Resonance in Medicine, 6,164-174, 1988).

The chelating ligands can also be bifunctional. That is, they can bindtightly to the metal ion forming a chelate while at the same timebearing a second functionality which confers upon it desirable chemical,physical and/or biological properties. Desirable physical properties ofthe chelator differ depending on the diagnostic or therapeutic purposeof the metal chelate. Desirable physical properties common to all usesare high affinity for the metal ion bound to the chelator, and ease ofsynthesis. When it is desired to use the metal chelate as a contrastmedium for NMR imaging or general purpose x-ray imaging, the desirablephysical properties are high water solubility, high chemical stabilityand viscosity and osmolality of a formulated drug solution as close aspossible to those of human blood. Further, in the specific instance of aspin relaxation catalyst, the greatest possible relaxivity is desired.Relaxivity as used herein is understood to be as the effectiveness, permole of complex, of altering the relaxation times of the nuclei beingimaged.

Human blood has an osmolality of 0.3 Osmol/kg-water. Hyperosmolality isa well known contributor to adverse patient reactions to injectedcontrast media, and the lower osmolality of newer x-ray agents is due totheir being nonionic molecules (possessing a net zero overall charge)(Shehadi, W. H.; “Contrast media adverse reactions: occurrence,reoccurrence and distribution patterns”, Radiol, 1982, 143, 11-17.Bettman, M. A.; “Angiographic contrast agents; conventional and newmedia compared”, Am. J. Roentgen, 1982, 139, 787-794. Bettman, M. A. andMorris, T. W.; Recent advances in contrast agents, Radiol. Clin. NorthAm., 1986, 24, 347-357.). Many gadolinium-based NMR agents in the priorart that are useful have a net negative overall charge, and thereforetheir aqueous formulated solutions have high osmolality. For example,Gd(DTPA)²-where DTPA stands for diethylenetriaminepentaacetic acid isformulated for use at 0.5M in water as the N-methylglucamine salt. Theosmolality of the solution is 1.6 to 2.0 Osmol/kg-water. New nonionic Gdcomplexes are described in U.S. Pat. Nos. 4,859,451 and 4,687,659. Thepreferred new gadolinium complexes of the present invention arenonionic—they are not salts. When these nonionic gadolinium complexesare formulated at 0.5M in water the osmolality of the solutions is0.3-0.6 Osmol/kg-water. The complex should be generally inert tointeraction with the body other than general tissue distribution andexcretion, usually by the renal route, without, or minimally, depositingGd metal in tissues for long periods of time. Gd complexes ofmacrocyclic aminocarboxylates are generally more chemically inert thanGd complexes of linear aminocarboxylates (P. Wedeking and M. Tweedle.Nucl. Med. Biol., 15, 395-402, 1988; M. Tweedle et al., Magn. Reson.Imog., 9, 409-415, 1991; and M. Tweedle, “Contrast and Contrast Agentsin Magnetic Resonance Imaging”, edited by P. A. Rink, European Workshopon Magnetic Resonance in Medicine, 1989) The preferred aminocarboxylateligands for Gd are therefore members of the macrocyclic aminocarboxylateclass, and are, in addition, nonionic. These properties are important toNMR imaging, but, in addition, the effectiveness of an agent for NMRimaging can be increased by altering the chemical structure so as toincrease the ability of the metal chelate to affect the relaxation timesof water protons.

In radiopharmaceutical imaging the doses administered are relativelysmall so that matching the drug formulation's physical properties tothose of human blood is relatively unimportant. In this use biologicalspecificity is more important. In particular, one could use ^(99m)Tc asthe metal and a chelating ligand which is functionalized with abiologically active entity such as a bile acid, fatty acid, amino acid,peptide, protein or one of numerous chemical entities known to bindreceptors in vivo. NMR contrast media may also make use of biologicalspecificity.

In radiopharmaceutical therapy, the metal ions may be chosen from amongthose known in the art; for example, ⁹⁰Y, ¹⁸⁸Re, ¹⁵³Sm. For this purposethe chelating ligand is generally covalently bound to a disease specificentity such as monoclonal antibody. When the metal-chelator-antibodyconjugate is injected into humans, it concentrates at the disease site,usually a malignant tumor. In this use the chelating ligand must containa reactive functionality which allows for a covalent bond to be formedbetween the chelating ligand and the antibody. Important characteristicsof the reactive functionality are as follows: (1) it must be covalentlyattached to the chelator such that it does not significantly diminishthe affinity of the chelator for the metal ion; (2) it must allow simplesynthesis in high yield of metal-chelator-antibody conjugates, theconjugate so-formed should have maximal affinity for its antigen, suchaffinity being minimally diminished as a result of covalently attachingthe metal-chelator; (3) it should ideally allow for rapid excretionand/or optimal dosimetry of the radioactive metal chelator in the eventthat the metal-chelator-antibody conjugate is decomposed or metabolizedin vivo.

When the metal is non-radioactive and paramagnetic such as gadolinium(III), the bifunctional chelate is useful in magnetic resonance imagingas a contrast agent, either as a discrete molecule or bound tosubstances such as lipids, sugars, alcohols, bile acids, fatty acids,receptor-binding ligands, amino acids, peptides, polypeptides, proteins,and monoclonal antibodies. When the metal is radioactive, such asyttrium(III) as ⁹⁰Y, the bifunctional chelate is useful in labelingmonoclonal antibodies for use in radiotherapy. When the metal is^(99m)Tc, ¹¹¹In, ²⁰¹Tl, ⁶⁷Ga, ⁶⁸Ga or the like, the chelate is useful inradiopharmaceutical imaging.

Two general methods have been employed for making bifunctional chelatesfrom chelating agents. In the first method one or more carboxylic acidgroups of a polyaminopolycarboxylic acid chelator are activated byconversion to such activating groups as internal or mixed anhydrides,activated esters (e.g., p-nitro phenyl, N-hydroxysuccinimide, etc.) orwith other derivatives known to those skilled in the art. The activatedacid group is then reacted with the protein. The metal ion is then addedto the protein-chelator complex.

There are two problems with this method. First, using a potential donorgroup, the carboxylic acid, to react with the protein can diminish thestrength of the chelate and contribute to the chemical lability of themetal ion. The second problem arises because the chelating ligands haveseveral carboxylates that are not uniquely reactive. When the chelatingligand is combined with an activating agent more than one species canresult because the number and chemical position of the groups activatedcannot be adequately controlled. When a mixture of such variouslyactivated chelating ligands is added to protein, protein-chelatorcomplexes of variable and uncertain chelating strength can be formed.Also, multiple activation of carboxylic acids on a chelator leads tointra- and inter-molecular crosslinking which is a major source ofdecreased immunospecificity. This problem could be overcome byseparating all of the products formed from the reaction of theactivating agent with the chelating ligand, but that process is verylaborious and makes the overall synthesis highly inefficient.

The second method for making a bifunctional chelate is to prepare achelating ligand with a unique reactive function, such as anisothiocyanate, attached to the chelating ligand at a position that doesnot substantially diminish the strength with which the chelating ligandbinds the metal ion. An article entitled “Synthesis of1-(p-isothiocyanato-benzyl) derivatives of DTPA and EDTA, AntibodyLabeling and Tumor-Imaging Studies” by Martin W. Brechbiel, Otto A.Gansow, Robert W. Atcher, Jeffrey Schlom, Jose Esteban, Diane E.Simpson, David Colcher, Inorganic Chemistry, 1986, 25, 2772 isillustrative of the above second method. Also, U.S. Pat. No. 4,885,363describes these methods as they apply specifically to nonionicmacrocyclic aminocarboxylates.

Wedeking et al., “Biodistribution and Excretion of New Gd-Complexes inMice”, Abstracts of the 8th Annual Meeting of the Society of MagneticResonance in Medicine, 801, 1989, have disclosed the compound

When used to chelate a paramagnetic ion, e.g., Gd, in magnetic resonanceimaging, this compound was found to have poor water solubility, althoughacceptable relaxivity.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to provide new metal-chelatingligands.

It is an object of this invention to provide new metal chelate complexesthat are nonionic.

Another object is to provide metal chelating ligands which whencomplexed with a metal heavier than iodine (e.g., Ba, Ta, Pb, Bi,Lanthanides) are effective as x-ray contrast agents.

Another object is to provide metal chelating ligands which whencomplexed with gamma emitting radioactive nuclide (e.g., ^(99m)Tc or¹¹¹In) are effective as imaging radiopharmaceuticals.

Another object is to provide metal chelating ligands which whencomplexed with beta or alpha emitting radioactive nuclide (e.g., ⁹⁰Y,¹⁵³Sm, ¹⁸⁸Re, ²¹²Bi) are effective as therapeutic radiopharmaceuticals.

It is a further object of this invention to provide metal-chelatingligands whose metal chelate complexes in aqueous solution have lowosmolality.

It is a further object of this invention to provide metal-chelatingligands whose metal chelate complexes have low acute toxicity.

It is a further object of this invention to provide metal-chelatingligands which, when complexed with a paramagnetic metal atom, areeffective as relaxation catalysts in magnetic resonance imaging.

It is a further object of this invention to provide bifunctionalmetal-chelating ligands that have the ability to covalently bind toproteins or other biologically active molecules thereby impartingbiological specificity to the metal chelate complex. The conversion ofthe novel molecules described herein to bifunctional chelates isaccomplished using the methods described above.

It is a further object of this invention to provide new metal complexeswith increased relaxivity.

It is a further object of this invention to provide bifunctionalmetal-chelating ligands that are thermodynamically stable, kineticallyinert and, when desired, electrically neutral.

These, and other objects which will be appreciated by the practitionerof this invention, are achieved by substituting at one of the nitrogenatoms of an aminocarboxylate ligand a substituted aromatic amide moietyof the formula

wherein

A₁ is —(CH₂)_(m)′— or a single bond;

(CH₂)_(m) and (CH₂)_(m)′ may independently be substituted with alkyl orhydroxyalkyl;

R₁₃ is hydrogen, alkyl, arylalkyl, aryl, alkoxy, hydroxyalkyl;

R₁ and R₂ are each independently hydrogen, alkyl, —NO₂, —NH₂,

NCS,

NR₃COR₉, where R₉ is alkyl or hydroxyalkyl, with the proviso that atleast one of R₁ and R₂ must be other than hydrogen;

R₃ and R₄ are independently hydrogen, alkyl, arylalkyl, aryl, alkoxy andhydroxyalkyl;

R₁₂ is hydrogen, alkyl or hydroxyalkyl;

m and m′ are independently 1 to 5;

and multimeric forms thereof.

Preferred are those compounds where A₁ is a single bond.

DETAILED DESCRIPTION OF THE INVENTION

The terms “alkyl” and “alkoxy” as used throughout the specification,refer to both straight and branched chain groups. Those groups having 1to 5 carbon atoms are preferred and methyl is the most preferred alkylgroup.

The term “aryl” as used throughout the specification refers to phenyland substituted phenyl. Preferred substituted phenyl groups are thosesubstituted with 1, 2 or 3 halogen, hydroxyl, hydroxyalkyl, alkyl,alkoxy, carbamoyl, carboxamide, acylamino or carboxyl groups.

Hydroxyalkyl refers to straight and branched alkyl bearing radicals R—OHgroups such as —CH₂CH₂OH, —CH₂CHOHCH₂OH, CH(CH₂OH)₂ and the like. Suchchemistry is well known to those skilled in the art (Sovak, M., editor,Radiocontrast Agents, Springer-Verlag, 1984, pp. 1-125).

As described above, aminocarboxylate nuclei known in the art can beprovided with a substituted aromatic amide moiety of formula I toprovide the novel compounds of the present invention.

Exemplary novel aminocarboxylates having a substituted aromatic amidemoiety include compounds of the formula

wherein in formulae Ia, Ib, Ic and Id, m, R₁₃, A₁, R₁, R₂ and R₁₂ are asdefined above for formula I and further wherein

X₁ is —COOY₁, PO₃HY₁ or —CONHOY₁;

Y₁ is a hydrogen atom, a metal ion equivalent and/or a physiologicallybiocompatible cation of an inorganic or organic base or amino acid;

A₂ is —CHR₆—CHR₇—, —CH₂CH₂(ZCH₂—CH₂)_(n)—,

wherein X₁ is as defined above;

each R₅ is hydrogen or methyl;

R₆ and R₇ together represent a trimethylene group or a tetramethylenegroup or individually are hydrogen atoms, lower alkyl groups (e.g., 1-8carbons), phenyl groups, benzyl groups or R₆ is a hydrogen atom and R₇is —(CH₂)_(p)—C₆H₄—W-protein where p is 0 or 1, W is —NH—, —NHCOCH₂— or—NHCS—, protein represents a protein residue;

n is 1, 2 or 3;

Z is an oxygen atom or a sulfur atom or the group NCH₂X₁ or NCH₂CH₂OR₈wherein X₁ is as defined above and R₈ is C₁₋₈alkyl;

V is X₁ or is —CH₂OH, —CONH(CH₂)_(r)X₁ or —COB, wherein X₁ is as definedabove, B is a protein or lipid residue, r is an integer from 1 to 12, orif R₅, R₆ and R₇ are each hydrogen; then both V's together form thegroup

where X₁ is as above, w is 1, 2 or 3, provided that at least two of thesubstituents Y₁ represent metal ion equivalents of an element with anatomic number of 21 to 29, 42, 44 or 57 to 83; from 1 to 4,advantageously 2 or 3, and preferably 3 M's are —OH and the balanceindependently are —OR₁₀, —NH₂, —NHR₁₀ and/or NR₁₀R₁₀′ wherein R₁₀ andR₁₀′ are selected from an organic alkyl radical of up to 18 carbon atomswhich may be substituted.

The compounds of formulae Ia, Ib, Ic and Id and salts thereof, can becomplexed with a para-magnetic metal atom and used as relaxationenhancement agents for magnetic resonance imaging. These agents, whenadministered to a mammalian host (e.g., humans) distribute in variousconcentrations to different tissues, and catalyze relaxation of protons(in the tissues) that have been excited by the absorption ofradiofrequency energy from a magnetic resonance imager. Thisacceleration of the rate of relaxation of the excited protons providesfor an image of different contrast when the host is scanned with amagnetic resonance imager. The magnetic resonance imager is used torecord images at various times generally before and after administrationof the agents, and the differences in the images created by the agents'presence in tissues are used in diagnosis. In proton magnetic resonanceimaging, paramagnetic metal atoms such as gadolinium(III), andoctahedral manganese(II), chromium(III) and iron(III) (all areparamagnetic metal atoms with a symmetrical electronic configuration)are preferred as metals complexed by the ligands of formula I;gadolinium(III) is most preferred due to the fact that it has thehighest paramagnetism, low toxicity, when complexed to a suitableligand, and high lability of coordinated water.

The metal-chelating ligands of the present invention can be complexedwith a lanthanide (atomic number 58 to 71) and used as chemical shiftagents in magnetic resonance imaging or in magnetic resonance in vivospectroscopy.

While the above-described uses for the metal-chelating ligands of thepresent invention are preferred, those working in the diagnostic artswill appreciate that the ligands can also be complexed with theappropriate metals and used as contrast agents in x-ray imaging,radionuclide imaging and ultrasound imaging.

Use in Imaging

To use the ligands of this invention for imaging, they must first becomplexed with the appropriate metal. This can be accomplished bymethodology known in the art. For example, the metal can be added towater in the form of an oxide or in the form of a halide or acetate andtreated with an equimolar amount of a ligand of the present invention.The ligand can be added as an aqueous solution or suspension. Diluteacid or base can be added (if needed) to maintain a neutral pH. Heatingat temperatures as high as 100° C. for periods up to four hours issometimes required, depending on the metal and the chelator, and theirconcentrations.

Pharmaceutically acceptable salts of the metal complexes of the ligandsof this invention are also useful as imaging agents. They can beprepared by using a base (e.g., an alkali metal hydroxide, meglumine orarginine) to neutralize the above-prepared metal complexes while theyare still in solution. Some of the metal complexes are formallyuncharged and do not need cations as counterions. Such neutral complexesare preferred as intravenously administered x-ray and NMR imaging agentsover charged complexes because they provide solutions of greaterphysiologic tolerance due to their lower osmolality.

Sterile aqueous solutions of the chelate complexes can be administeredto mammals (e.g., humans) orally, intrathecally and especiallyintravenously in concentrations of 0.003 to 1.0 molar. For example, forthe visualization of brain lesions in canines using magnetic resonanceimaging, a gadolinium complex of a ligand of formula I can beadminstered intravenously at a dose of 0.05 to 0.5 millimoles of thecomplex per kilogram of animal body weight, preferably at a dose of 0.1to 0.3 millimoles/kilogram. For visualization of the kidneys, the doseis preferably 0.05 to 0.25 millimoles/kilogram. For visualization of theheart, the dose is preferably 0.25 to 1.0 millimoles/kilogram. The pH ofthe formulation will be between about 6.0 and 8.0, preferably betweenabout 6.5 and 7.5. Physiologically acceptable buffers (e.g.,tris-(hydroxymethyl)aminomethane) and other physiologically acceptableadditives (e.g., stabilizers such as parabens) can be present.

It is also advantageous to employ dual scavenging excipients such asthose described in a copending application U.S. Ser. No. 682,487 filedApr. 9, 1991 entitled “DUAL FUNCTIONING EXCIPIENT FOR METAL CHELATECONTRAST AGENTS”. Those excipients have the general formulaX_(m)[X′(L′)]_(n)wherein X and X′ are independently Ca or Zn, L′ is an organic ligandwhich may be different than or the same as the ligand employed tocomplex the metal and m and n are independently 1, 2 or 3.Use of Radiotherapy or Imaging where the Metal-Chelate-Complex is Boundto a Biomolecule

The bifunctional metal-chelating ligands can bind to a monoclonalantibody or a fragment thereof for use in radiotherapy. Monoclonalantibodies are useful in that they can be used to target radio-nuclidesto cancer or tumor sites with great specificity. The compounds of thisinvention wherein R₁ is other than hydrogen are then linked tomonoclonal antibodies or fragments thereof.

The methods of linking the bifunctional chelate to the antibody orantibody fragment are known in the art (Brechbiel, same reference asreferred to hereinabove) and will depend primarily on the particularbifunctional chelate and secondarily on the antibody or fragmentthereof. For example, when the formula Ia compound is R₁=H, R₂=—NCS or

one reacts 10 μL of a 5.0 mM aqueous solution of the formula I chelatorwith 0.5 mL of a 5.0 mg/mL monoclonal antibody (B72.3 purchaseable fromDamon Biotech Corporation) in 50 mM Hepes buffer at pH 8.5. 16 μL of1.5M aqueous triethylamine is added. After 2 hours reaction time, themonoclonal antibody is purified by dialysis. This procedure providesbetween 1 and 2 formula I chelator molecules bound to each monoclonalantibody. Radioactive metal ion (for example ⁹⁰Y) can then be added tothe monoclonal antibody-bound chelator by methods known in the art. Forexample, ⁹⁰Y as the ⁹⁰Y(III) (acetate)₃(H₂O)₄ (approximate formula inaqueous solution) can be reacted with the monoclonal antibody-boundchelate in solutions where the concentration of each is between 10⁻⁵ and10⁻⁷ and the pH is 6. Dialysis against citrate is then used to purifythe product.

An alternative, and preferred method follows that described above, butsubstitutes the metal-chelate complex for the chelating ligand. To usethis method the metal chelate complex is first made by reactingmetal-oxide, -halide, -nitrate, -acetate, or the like with formula Ichelator. For the chelator described above the acetate of ⁹⁰Y at <10⁻⁶Mis reacted with the chelator at about 10⁻³ at pH 6, the chelate complexis purified by ion exchange or reverse phase HPLC choromatography, andthen reacted with the monoclonal antibody described above for thechelator. The bifunctional, metal-containing, linked antibody is used inthe following manner. A human or animal with a tumor to which themonoclonal antibody is specific is injected intravenously,subcutaneously, intraperitoneally or intralymphatically for example,with an aqueous solution of the ⁹⁰Y-formula I chelator-monoclonalantibody compound. This allows the radioactive metal ion to be directedto the tumor for which it is intended. The intravenous dosage used is0.1 to 0.4 millicuries per kilogram of body weight.

Preferred embodiments for when the compounds are linked to a protein arewhen R₁ and/or R₂=NCS is reacted with protein to produce the proteinconjugate. Preferred proteins are those in serum, wherein the R₁ and/orR₂=—NCS compound is directly injected.

It is understood that other functional groups known in the art can beused to link the bifunctional metal-chelating ligands of this inventionto monoclonal antibodies or fragments thereof.

R₁ and R₂ are each

and R₃ in each is hydroxyalkyl in a preferred embodiment for forming aGd(III) chelate useful in general purpose magnetic resonance imaging.The most preferred embodiments for forming a Gd(III) chelate are whenthe R₃ groups are each

or —CH(CH₂OH)₂, especially

and the R₄ groups are each hydrogen.

The present invention also includes multimeric forms of the compounds offormula I, such as dimers, trimers, tetramers, etc. Known functionalgroups and technology as those discussed above regarding conjugationwith biomolecules are readily useable to provide such multimers. Thefunctional groups provided onto the phenyl ring

can be, for example, R₂=NCS or

especially where R₁₂ is methyl or ethyl. Thus, exemplary multimers offormula I

where Q is the aminocarboxylate nucleus of Ia, Ib, Ic or Id, are shownby

and the like,

Preparation of Formulae Ia, Ib, Ic and Id Compounds

To prepare the compounds of formula Ia, a compound of the formula

is reacted in a solvent, e.g., water, and in the presence of a base,e.g., sodium hydroxide, with a compound of the formula

wherein L is a leaving group, such as halogen. The preparation ofcompounds of formula II is well known, for example, in U.S. Pat. No.4,885,363 to Tweedle et al. For example, in preparing compounds offormula II, reaction of a compound of the formula

where Y is

with a compound of the formula

wherein L is a leaving group such as halogen is preferably carried outin water at a pH of about 8.5 to 11 and the temperature of the reactionis maintained at about 45°-55° C. Preferably, only about two equivalentsof a compound of formula v are initially used in the reaction; anadditional equivalent of the compound of formula V is added in portionsstarting about 2 to 3 hours after the reaction begins. Total reactiontime will preferably be about 8 to 24 hours. The desired trisubstitutedproduct can be separated from the reaction mixture, which includes themono-, di-, tri- and tetra-substituted derivatives, by techniquesrecognized in the art including selective precipitation, chromatographyand crystallization.

A preferred preparation of the compounds of formula IIa wherein R₁₂ ishydrogen is to react 1,4,7,10-tetraazacyclododecane, known in the art,with dimethylformamidedimethylacetal in the presence of benzene to yield1,4,7,10-tetraazatricyclo-[5.5,1.0]tridecane. This “tricyclic” compoundis reacted with an ethanol/water mixture to yield1-formyl-1,4,7,10-tetraazacyclododecane. This formyl compound is thenreacted with t-butyl bromoacetate to yield1-formyl-4,7,10-triscarboxymethyl-1,4,7,10-tetraazacyclododecane,tris-t-butylester. Finally, the ester groups are removed in the presenceof strong acid, such as sulfuric acid, to yield a compound of formulaIIa wherein R₁₂ is hydrogen. The most preferred methods are included inDischino, et al., Inorg. Chem., 30, 1265, 1991.

Compounds of formula III wherein R₁ and R₂ are each

and R₁₃ is hydrogen are prepared by first reacting a compound of theformula

in a solvent, e.g., methanol, with a compound of the formulaH₂NR₃   VIIto provide the intermediate

Compounds of formula VIII can thereafter be reduced, e.g., with hydrogenin the presence of a palladium on carbon catalyst, to provide

Reaction of compound IX with a compound of the formula

wherein L and L′ are the same or different leaving groups, e.g.,halogens, in a solvent, e.g., dimethylacetamide, provides the compoundsof the formula

that is, compounds of formula III where R₁ and R₂ are each

A₁ is a single bond, and R₁₃ is hydrogen.

In the event that the R₃ group in intermediate VIII containshydroxyalkyl moieties, the hydroxy groups are converted to acetyloxygroups after the reaction of compounds VI and VII to obtain VIII′. Forexample, if the compound of formula VII is

reaction as described above with compound VI provides

wherein Ac is acetyl. Following reduction to the corresponding anilineand thereafter reaction with compound X, the corresponding intermediatesof III′ i.e., wherein R₃ is acetyloxy alkyl, are converted to theirhydroxyalkyl counterparts by known treatment, e.g., with sodiummethoxide in a solvent, for example, methanol.

Compounds of formula III wherein A₁ is —CH₂—, R₁₃ is as defined above, Lis chloro and R₁ and R₂ are each

are prepared by first reacting a compound of the formula

with gaseous hydrogen in the presence of a catalyst such as palladium oncarbon in dilute mineral acid to provide the aniline

The aniline is diazotized with nitrous acid in acidic medium and thentreated with sodium cyanide to obtain

Reduction of the nitrile XI in the presence of a platinum catalyst withgaseous hydrogen at low pressure, for example 3 atmospheres, affords

Reaction of XII with a compound of formula

wherein L is a leaving group, for example chlorine, provides compoundsof the formula

If R₁₃ is other than hydrogen, for example methyl, the compound of theformula XII is treated with an aldehyde R₈CHO, for example formaldehyde,wherein R₈ is H, under reducing conditions, for example with sodiumborohydride, to obtain

wherein R₁₃ is methyl. Reaction of IX′ with the chloride of formula XIIIwill provide the desired intermediate of the formula

Similarly, compounds of formulae Ib, Ic and Id can be prepared byreacting the various compounds of formula III in a solvent, e.g. water,and in the presence of a base, e.g., sodium hydroxide with thecorresponding compounds

Compounds of formula IIb and IIc are described in U.S. Pat. No.4,647,447. Compounds of formula IId are described in U.S. Pat. No.4,859,451.

The invention will now be further described by the following examples,but is not limited to the details therein.

EXAMPLE 110-[2-[[3,5-Bis[[(2,3-dihydroxypropyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium complex A.N,N′-Bis[2,3-bis(acetyloxy)propyl]-5-nitro-1,3-benzenedicarboxamide

To a solution of dimethyl-5-nitroisophthalate (23.9 g, 100 mmol) inmethanol (300 mL) was added 1-amino-2,3-propanediol (20.2 g) and themixture was refluxed for 48 hours. Methanol was removed in vacuo, theresidue was dissolved in pyridine (150 mL) and then treated with aceticanhydride (80 mL) at room temperature for 16 hours. Excess aceticanhydride was decomposed by adding water (50 mL) to the reactionmixture. The solvents were removed in vacuo, the residue dissolved inethyl acetate (400 mL) and washed with water (2×100 mL), 10%hydrochloric acid (200 mL) and finally with brine (100 mL). The ethylacetate layer was dried and removal of the solvent afforded the title Anitro-bis-amide (51.8 g), as a light yellow viscous syrupy material.This was used directly without further purification in the next step.

B. 5-Amino-N,N′-bis[2,3-bis(acetyloxy)propyl]-1,3-benzenedicarboxamide

A solution of the title A nitrobisamide (31.5 g, 60 mmol) in methanol(180 mL) was hydrogenated over 10% palladium on carbon (300 mg) for aperiod of 3 hours. The catalyst was filtered off and the solvent removedin vacuo to afford pure title B aniline (28.6 g), as a viscous syrupymaterial. This was used directly without further purification in thenext step.

C.N,N′-Bis[2,3-bis(acetyloxy)propyl]-5-N-[(chloroacetyl)amino]-1,3-benzenedicarboxamide

The title B aniline was dissolved in dimethylacetamide (150 mL) andtreated with chloroacetyl chloride (11.28 g, 100 mmol) dropwise over aperiod of 20 minutes. The solution was stirred for 3 hours anddimethylacetamide removed in vacuo. The residue that resulted wasdissolved in ethyl acetate, washed with water, 10% aqueous sodiumbicarbonate, and finally with water. The ethyl acetate layer was driedand removal of the solvent afforded the crude chloroacetanilide (32.0g). The crude material was purified by column chromatography over silicagel to obtain the title C compound (26.3 g), as a colorless glassysolid. An analytical sample was prepared by crystallizing 1.00 g of theglassy solid from ethyl acetate/hexane. m.p. ______ (68°-72°)

Elemental Analysis calc'd for C₂₄H₃₀N₃ClO₁₁: C, 50.40; H, 5.29; N, 7.35;Cl, 6.20; O, 30.77%. Found: C, 50.28; H, 5.15; N, 7.11; Cl, 6.25%.

D.5-[(Chloroacetyl)amino-N,N′-bis[2,3-dihydroxypropyl)-1,3-benzenedicarboxamide

A solution of the title C compound (25.6 g, 45 mmol) in methanol (200mL) was treated with sodium methoxide (20 mmol) and the solution wasstirred at 0° for 30 minutes. The pH of the reaction mixture wasadjusted to 7 by adding Dowex 50 (H⁺) resin, the resin filtered off andthe methanol removed in vacuo to afford pure title D compound as acolorless glassy solid (16.8 g). This material was directly used in thenext step without further purification.

E.10-[2-[[3,5-Bis[[(2,3-dihydroxypropyl)amino]-carbonyl]phenyl]amino]2-oxoethyl]1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

A solution of DO3A sulfate(DO3A=1,4,7,-triscarboxymethyl-1,4,7,10-tetraazacyclododecane preparedin U.S. Pat. No. 4,885,363 to Tweedle et al.) (12.0 g, 27 mmol) was madein water (80 mL) and the pH of the solution was adjusted to 9.8 byadding 5 M sodium hydroxide. While maintaining the pH of the solution at9.8, a solution of the title D compound (16.4 g, 40.6 mmol) in water (50mL) was slowly added to the DO3A solution at 80° over a period of 45minutes. At the end of 17 hours, the reaction mixture was cooled to roomtemperature, the pH lowered to 3.5 by adding 1 N hydrochloric acid andthe solution was desalted by cation exchange chromatography. Furtherpurification by anion exchange chromatography afforded the title Ecompound as the triethylammonium salt (19.9 g). The triethylamine saltwas dissolved (6.00 g) in water (1 L), applied to an anion exchangecolumn and then eluted with 50 mM formic acid tc obtain the desiredtitle E compound (4.9 g). IR: 3400 (OH); 3115 (NH); 1631 (COOH andArCONH) cm⁻¹. Mass Spectrum: 714 (M+H)⁺; 712 (M−H)⁻.

Elemental Analysis calc'd for C₃₀H₄₇N₇O₁₃.0.38H₂O: C, 50.01; H, 6.68; N,13.61; O, 29.71%. Found: C, 49.91; H, 6.97; N, 13.42; H₂O, 0.95.%.

F.10-[2-[[3,5-Bis[[(2,3-dihydroxypropyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

To a solution of the title E triethylammonium salt (19.00 g, 18.7 mmol)in water (80 mL) at pH 4.72 was added a solution of Gd(OAc)₃.4H₂O (9.83g, 24 mmol) in water (80 mL) and the reaction mixture was stirred atroom temperature for 12 hours. The reaction mixture was subjected to lowpressure reversed phase column chromatography over astyrene-divinylbenzene copolymer resin to obtain the title compound as acolorless glassy solid (17.5 g). The pure product (17.00 g) wascrystallized from hot methanol to afford this title F compound ascolorless needles of >99.9% purity. This sample was redissolved in water(200 mL), the solvent removed, and the sample dried in vacuo (1 mm) forfour days at 80°. Mass Spectrum: 869 (M+H)⁺, 867 (M−H)⁻¹.

Elemental Analysis: calc'd for C₃₀H₄₄N₇O₁₃.0.36 H₂O: C, 41.20; H, 5.15;N, 11.21; O, 24.45%. Found: C, 40.96; H, 5.07; N, 10.93; H₂O, 0.75.%.

EXAMPLE 210-[2-[[3,5-Bis-[[[2-hydroxy-1-(hydroxymethyl)ethyl]-amino]carbonyl]phenylamino]2-oxoethyl]1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid A.N,N′-bis[2-(Acetyloxy)-1-[(acetyloxy)methyl]-ethyl]-5-nitro-1,3-benzenedicarboxamide

To a solution of dimethyl-5-nitroisophthalate (14.0 g, 58 mmol) inmethanol (150 mL) was added 2-amino-1,3-propanediol (16.5 g, 181 mmol)and the mixture was refluxed for 48 hours. The reaction mixture wascooled to room temperature and the crystalline solid that separated outfiltered and dried to obtain the bis amide (19.5 g). A solution of thebis-amide (19.0 g) in pyridine (75 mL) was treated with acetic anhydride(40 mL) at room temperature for 16 hours. Excess acetic anhydride wasdecomposed by adding water (50 mL) to the reaction mixture. The solventswere removed in vacuo, the residue dissolved in ethyl acetate, and thesolution washed with water, 10% hydrochloric acid and finally withbrine. The ethyl acetate layer was dried and removal of the solventafforded pure title A nitrobisamide (23.6 g) as a colorless solid, aftercrystallization from acetone and hexane, m.p. 105-107° C.

B.5-Amino-N,N′-bis[2-(acetyloxy)-1-[(acetyloxy)-methyl]ethyl]-1,3-benzenedicarboxamide

A solution of the title A compound (18.0 g, 34 mmol) in methanol (180mL) was hydrogenated over palladium on carbon (0.5 g) for a period of 3hours. The catalyst was filtered off and the solvent removed in vacuo toafford pure title B aniline (16.6 g) after crystallization from acetoneand hexane, m.p. 152-154° C.

Elemental Analysis calc'd for C₂₄H₃₀N₃ClO₁₁: C, 50.40; H, 5.29; N, 7.35;Cl, 6.20%. Found: C, 50.64; H, 5.20; N, 7.22; Cl, 6.57.%.

C.N,N′-Bis[2-(acetyloxy)-1-[(acetyloxy)methyl]-ethyl]-5-(chloroacetyl)amino-1,3-benzenedicarboxamide

The title B compound (17.0 g, 34 mmol) was dissolved indimethylacetamide (150 mL) and treated with chloroacetyl chloride (7.52g, 64 mmol) dropwise over a period of 20 minutes. The solution wasstirred for 3 hours and dimethylacetamide was then removed in vacuo. Theresidue that resulted was dissolved in ethyl acetate, washed with water,saturated aqueous sodium bicarbonate solution, and finally with water.The ethyl acetate layer was dried and the solvent removed to obtain thecrude chloroacetanilide (18.5 g). This material was crystallized fromethyl acetate and hexane to afford pure title C compound (16.8 g), m.p.135-137° C.

Elemental Analysis calc'd for C₂₄H₃₀N₃ClO₁₁: C, 50.40; H, 5.29; N, 7.35;Cl, 6.20; O, 30.77%. Found: C, 50.64; H, 5.20; N, 7.22; Cl, 6.57%.

D.5-[(Chloroacetyl)amino]-N,N′-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-1,3-benzenedicarboxamide

A solution of the title C compound (16.0 g, 28 mmol) in methanol (200mL) was treated with sodium methoxide (10 mmol) and the solution stirredat 0° for 30 minutes. The precipitated solid was filtered and dried toafford pure title D compound as a colorless glassy solid (10.8 g), m.p.222-224°. Mass Spectrum: m/z 404 (M+H)⁺.

Elemental analysis calc'd for C₁₆H₂₂N₃ClO₇: C, 47.59; H, 5.49; N, 10.41;Cl, 8.78; O, 27.73%. Found: C, 47.66; H, 5.55; N, 9.98; Cl, 8.88%.

E.10-[2-[[3,5-Bis-[[[2-hydroxy-1-(hydroxy-methyl)ethyl]amino]carbonyl]phenylamino]2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

A solution of DO3A sulfate (6.0 g, 13.5 mmol) was made in water and thepH of the solution was adjusted to 9.8 by adding 5 M sodium hydroxide.While maintaining the pH of the solution at 9.8, solidN,N′-bis[2-hydroxy-1-[(hydroxy)methyl]ethyl]-5-N-(chloroacetyl)aminobenzene-1,3-dicarboxamide(8.2 g, 20.4 mmol) was added in small portions to the DO3A solution at80° over a period of 45 minutes. At the end of 20 hours, the reactionmixture was cooled to room temperature, the pH lowered to 3.5 by adding1 N hydrochloric acid and the solution was desalted by cation exchangecolumn chromatography. Further purification by anion exchange columnchromatography afforded the title E compound as the correspondingtriethylammonium salt (5.2 g). The triethylammonium salt (5.2 g) wasdissolved in water (1 L) and applied to an anion exchange column andeluted with 50 mM formic acid to obtain the pure title E compound (4.4g), as a colorless glassy solid.

Elemental analysis calc'd for C₃₀H₄₇N₇O₁₃: C, 50.48; H, 6.64; N, 13.74;O, 29.14%. Found: C, 50.34; H, 6.83; N, 13.54%.

F. The Gadolinium Chelate of Title E Compound

The Gd complex of this ligand was prepared by the same method used forthe compound in Example 1.

Elemental Analysis for C₃₀H₄₄N₇O₁₃Gd 3.48 H₂O: C, 38.72; H, 5.52; N,10.53; O, 28.33%. Found: C, 39.01; H, 5.37; N, 10.26%.

EXAMPLE 310-[2-[[3,5-Bis[[(2-methylbutyl)amino]carbonyl]-phenyl]amino]2-oxoethyl]1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium complex A.N,N′-Bis[(2-methylbutyl)amino]-5-nitro-1,3-benzenedicarboxamide

To a solution of dimethyl-5-nitro-isophthalate (14.0 g, 50 mmol) inmethanol was added 2-methylbutylamine (12.5 g, 150 mmol) and the mixturewas refluxed for 48 hours. Methanol was removed in vacuo, the residuedissolved in ethyl acetate and washed with 10% hydrochloric acid, 10%aqueous sodium bicarbonte solution and finally with water. The ethylacetate layer was dried and removal of the solvent afforded the desiredcompound. This was crystallized from ethyl acetate and hexane to affordthe the title A compound as colorless needles (19.6 g), m.p. 147-148° C.

Elemental Analysis for C, 61.87; H, 7.79; N, 12.03; O, 18.31%. Found: C,61.84, H, 7.76; N, 12.02%.

B. 5-Amino-N,N′-bis[(2-methylbutyl)amino]-1,3-benzenedicarboxamide

A solution of the title A compound (17.45 g, 50 mmol) in methanol (180mL) was hydrogenated over 10% palladium on carbon (500 mg) for a periodof 3 hours. The catalyst was filterd off and the solvent removed invacuo to afford the title aniline as a colorless solid. This wascrystallized from acetone and hexane to afford the title B compound ascolorless needles (15.8 g), m.p. 170-172° C.

Elemental Analysis for C, 67.87; H, 9:15; N, 13.15; O, 12.02%. Found: C,68.07, H, 9.30; N, 13.26%.

C.5-[(Chloroacetyl)amino]-N,N-bis[(2-methyl-butyl)amino]1,3-benzenedicarboxamide

A solution of the title B compound (11.48 g, 36 mmol) indimethylacetamide (200 mL) was treated with chloroacetyl chloride (5.6g, 50 mmol) dropwise over a period of 20 minutes. The solution wasstirred for 3 hours and dimethylacetamide removed in vacuo. The residuethat resulted was dissolved in ethyl acetate (200 mL), washed with water(100 mL), 10% aqueous sodium bicarboante (100 mL) and finally with water(100 mL). The ethyl acetate layer was dried and removal of the solventafforded the crude chloroacetanilide (12.8 g). This was crystallizedfrom ethyl acetate and hexane to afford the title C compound ascolorless needles (11.2 g), m.p. 160-162° C.

Elemental Analysis for C, 60.67; H, 7.64; N, 10.61; Cl, 8.95; O, 12.12%.Found: C, 61.03, H, 7.69; N, 10.57; Cl, 9.22%.

D.10-[2-[[3,5-Bis[[(2-methylbutyl)amino)-carbonyl]phenyl]amino]2-oxoethyl]1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

A solution of DO3A sulfate (6.0 g, 13.5 mmol) was made in water (100 mL)and the pH of the solution was adjusted to 9.8 by adding 5 M sodiumhydroxide. While maintaining the pH of the solution at 9.8, a solutionof the chloroacetanilide (8.2 g, 27 mmol) in ethanol (100 mL) was slowlyadded to the DO3A solution at 80° over a period of 1 hour. At the end of17 hours, the reaction mixture was cooled to room temperature, the pHlowered to 3.5 by adding 1N hydrochloric acid and the solution wasdesalted by cation exchange chromatography. Further purification byanion exchange chromatography afforded the title compound as thetriethyl ammonium salt (2.8 g). The triethyl ammonium salt was dissolvedin water, applied to an anion exchange column and then eluted with 50 mMformic acid to obtain the desired compound (AAA-DO3A) (2.2 g). A smallamount of an impurity present in this sample was further removed by areverse phase CHP-20 column chromatography to afford the title compoundas a colorless glassy solid (1.8 g). Mass Spectrum: 706 (M+H)⁺; 704(M−H)⁻.

Elemental analysis calc'd for C₃₄H₅₅N₇O₉.1.7 H₂O: C, 55.45; H, 7.99; N,13.31; O, 23.24. Found: C, 55.85; H, 8.37; N, 13.19; H₂O: 4.15.

E. Gadolinium Chelate of Title D Compound

The Gd complex of this ligand was prepared by the same method as usedfor the compound of Example 1.

Elemental anal. calc'd for C₃₄H₅₂N₇O₉Gd, 6.44 H₂O: C, 41.84; H, 6.70; N,10.04; O, 26.31%. Found: C, 41.80; H, 6.65; N, 10.28%.

The T₁ relaxivity was measured for nine prior art gadolinium complexes(1-9) as compared to novel gadolinium complexes using the ligands ofExamples 1, 2 and 3 (#10, 11 and 12, respectively, in the Table below).Relaxivity was measured on an IBM Minispec spin analyzer operating at 20MHz and 39±1° C. Aqueous solutions were used in 0.1-5 mM Gdconcentration range.

Structures of Ligands for Gd Complexes of Table 1 1. DTPA

2. DTPA-HA

3-12.

3. DOTA Y = O Y = O⁻ 4. NH₂-DO3A X = O Y = —NH₂ 5. MA-DO3A X = O Y =—NHCH₃ 6. HEA-DO3A X = O Y = —NHCH₂CH₂OH 7. PA-DO3A X = O Y = —NHPhenyl8. HP-DO3A X = OH, H Y = —CH₃ 9. PG-DO3A

10. HAA-DO3A X = O (Ex. 1)

11. HAS-DO3A X = O (Ex. 2)

12. AAA-DO3A X = O (Ex. 3)

TABLE 1 Data on Water Soluble Gd Complexes and Ions Demonstrating TheEnhancement of Relaxivity by N-Hydroxy-alkyl or N-alkyl- isophthalamideGroups and by Aryl Groups or by Hydroxyalkyl or Alkylamido Groups.Gd(L), L = T₁ Relaxivity  1. DTPA 3.7  2. DTPA-HA 4.4  3. DOTA 3.4  4.NH₂-DO3A 3.6  5. MA-DO3A 4.3  6. HEA-DO3A 4.3  7. PA-DO3A 4.1  8.HP-DO3A 3.7  9. PG-DO3A 3.4 10. HAA-DO3A (Ex. 1) 5.8 11. HAS-DO3A (Ex.2) 5.4 12. AAA-DO3A (Ex. 3) 5.9

The relaxivity is especially high only in the substituted arylcompounds, 10, 11 and 12, i.e., Gd(HAA-DO3A), Gd(HAS-DO3A) andGd(AAA-DO3A). One or two hydroxy groups alone do not enhance relaxivity,as can be seen from L=HP-DO3A, PG-DO3A. Alkyl or aryl substituents onlyslightly enhance relaxivity, as seen from MA-DO3A and PA-DO3A. Bothalkyl and hydroxyalkyl substituents on the aromatic are effective atenhancing relaxivity (the hydroxyalkyls are preferred for theirincreased water solubility).

EXAMPLE 410-[2-[Methyl[3,5-bis[[(2-methylbutyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt A.N,N′-bis(2-Methylbutyl)-5-[[(phenylmethoxy)-carbonyl]-amino]-1,3-benzenedicarboxamide

To a cooled solution of compound A from Example 3 (15.4 g, 46 mmol) inanhydrous DMA (75 ml) at 0° C. was added benzyl chloroformate (9.4 g,55.2 mmol). The clear solution was stirred at 0° C. for 2 hours. DMA wasremoved in vacuo. The residue was dissolved in EtOAc (150 ml), and waswashed with aqueous NaHCO₃ solution (30 ml) and with H₂O (2×50 ml). Theorganic layer was dried over anhydrous MgSO₄ and the solvent removed toobtain the crude product as an oily liquid. Recrystallization of thecrude material from EtOAc/hexanes afforded the title A product as awhite solid (17.0 g), m.p. 130.5-132.5° C.

Elemental analysis calc'd for C₂₆H₃₅N₃O₄: C, 68.85; H, 7.78; N, 9.26; O,14.11%. Found: C, 68.64; H, 7.91; N, 9.20%.

B.N,N′-bis(2-Methylbutyl)-5-[methyl[(phenyl-methoxy)carbonyl]amino]-1,3-benzenedicarboxamide

To a suspension of NaH (0.58 g, 24.2 mmol) in anhydrous THF (25 ml) wasadded a solution of the title A compound (10.0 g, 22 mmol) in anhydrousTHF (60 ml). MeI (15.7 g, 110 mmol) was added and the reaction mixturewas stirred at room temperature for 1 hour. THF was removed in vacuo.The solid was dissolved in EtOAc and washed with H₂O and then withaqueous NaCl solution. The EtOAc layer was dried over anhydrous MgSO₄and the solvent removed to obtain the title B compound.

Elemental analysis calc'd for C₂₇H₃₇N₃O₄: C, 69.35; H, 7.98; N, 8.99; O,13.69%. Found: C, 69.10; H, 8.03; N, 8.91%.

C. 5-(Methylamino)-N,N′-bis(2-methylbutyl)-1,3-benzenedicarboxamide

To a solution of the title B compound (13 g, 27.8 mmol) in MeOH (50 ml)was added 1,4-cyclohexadiene (20 ml) and 10% Pd/C (3.25 g). The mixturewas refluxed for 0.5 hour. The solid was filtered through a celite cakeand the solvent was removed to obtain the crude product.Recrystallization from hot EtOAc afforded the title C product as whitecrystals (5.2 g), m.p. 160.1-160.8° C.

Elemental analysis calc'd for C₁₉H₃₁N₃O₂: C, 68.43; H, 9.37; N, 12.60;O, 19:60%. Found: C, 68.13; H, 9.50; N, 12.57%.

D.5-[(Chloroacetyl)methylamino]-N,N′-bis(2-methylbutyl)-1,3-benzenedicarboxamide

To a solution of the title C compound (5.2 g, 15.6 mmol) in anhydrousDMA (150 ml) was added chloroacetyl chloride (2.43 g, 5.9 mmol). Thesolution was stirred at room temperature for 1.5 hours. The mixture wascooled. Water (20 ml) was added and the solvent removed in vacuo. Theresidue was dissolved in EtOAc and washed with aqueous NaHCO₃ solution,then with water. The organic layer was dried over anhydrous MgSO₄ andthe solvent removed to obtain the crude product. Recrystallization fromhot EtOAc afforded the title D compound as white crystals (6.0 g), m.p.170.0-171.5° C.

Elemental analysis calc'd for C₂₁H₃₂N₃O₃Cl: C, 61.53; H, 7.87; N, 10.25;Cl, 8.65; O, 11.71%. Found: C, 61.77; H, 7.83; N, 10.39; Cl, 8.41%.

E.10-[2-Methyl[3,5-bis[[(2-methylbutyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7-10-tetraazacyclododecane-1,4,7-triaceticacid

DO3A sulfate (4.35 g, 9.8 mmol) was dissolved in H₂O (100 ml) and the pHof the solution adjusted to 9.8 by adding 10 N NaOH. To this solution at85° C. was added a solution of the title D compound (5.7 g, 13.9 mmol)in EtOH (100 ml) over a period of 45 minutes. The pH was maintained at9.8 by adding 5 N NaOH. The mixture was heated at 85° C. for 44 hours.The solvents were removed in vacuo. The solid was dissolved in H₂O (300ml) and EtOAc (100 ml) and the cloudy solution was stirred at 85° C. for2 hours until the mixture turned clear. The two layers were separated.The aqueous layer (pH 7) which contained the crude product was appliedto a 300 ml column of CHP-20 resin using EtOH/H₂O (0-10%) as an eluent.The fractions containing the desired compound were combined and removalof the solvent afforded the title E product as a monosodium salt (2.9g).

Elemental analysis calc'd for C, 54.86; H, 7.68; N, 13.17; O, 19.76%.Found: C, 54.84; H, 8.09; N, 12.73%.

F.10-[2-[Methyl[[3,5-bis[[(2-methylbutyl)amino]-carbonyl]phenyl[amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

The title E compound (700 mg, 0.97 mmol) was dissolved in H2O (8 ml) andthe pH of the solution adjusted to 4.5 by adding diluted HOAc. To thissolution was added a solution of Gd(OAc)₃.4H₂O (1.21 g, 1.3 mmol) in H₂O(10 ml). The mixture was stirred at 45° C. for 24 hours. The solutionwas then applied to a 600 ml column of CHP-20 resin, using EtOH/H₂O(0-50%) as an eluent. The fractions containing the desired compound werecombined and removal of the solvent afforded 770 mg of the titleproduct.

Elemental analysis calc'd for C₃₄H₅₂N₇O₉Gd.1.10H₂O: C, 46.42; H, 6.21;N, 11.14; Gd, 18.28; O, 16.74%. Found: C, 46.68; H, 6.35; N, 10.88%.

EXAMPLE 510-[2-[[4-[[2,3-Dihydroxypropyl)amino]carbonyl]-phenyl]amino]-2-oxoethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt A.N-(2,3-Diacetylpropyl)-4-carboxyamidonitrobenzene

To a solution of methyl 4-nitro benzoate (18.1 g, 100 mmol) in 200 ml ofMeOH was added 3-amino-1,2-propanediol (18.2 g, 200 mmol) and themixture was refluxed for 24 hours. The product then was directlyacetylated. Methanol was removed in vacuo. The residue was dissolved in100 ml of pyridine and 80 ml of acetic anhydride was added. The solutionwas stirred at room temperature for 24 hours. The solution was cooledand water was added to decompose the excess acetic anhydride. Thesolvents were removed in vacuo. The residue was dissolved in EtOAc andit was washed with H₂O, 10% HCl and finally with brine. The organiclayer was dried and removal of the solvent afforded 28.3 g of the titleA compound as a yellowish solid (87.3 mmol), m.p. 101.5-102.8°.

Elemental analysis calc'd for C₁₄H₁₆N₂O₇: C, 51.85; H, 4.97; N, 8.64.Found: C, 51.69; H, 5.00; N, 8.58.

B. N-(2,3-Diacetylpropyl)-4-carboxyamido aniline

A solution of the title A compound (12 g, 37 mmol) in 120 ml of EtOAcwas mixed with 5% Pd/c (1.2 g). The solution was hydrogenated at 45 psipressure until the pressure dropped down to a constant value. The solidwas then filtered. The filtrate was concentrated to dryness and 10.8 gof the title B product as a foaming liquid was obtained (36.7 mmol).TLC: Silica gel, R_(f) 0.70, EtOAc, visualized by UV.

C. 4[(Chloroacetyl)amino]-N-(2,3-dihydroxy-propyl)-1-benzenecarboxamide

To a cooled solution of the title B compound (9.3 g, 31.6 mmol) in 120ml of anhydrous DMA was added chloroactyl chloride (5.3 g, 46.9 mmol).The solution was stirred at room temperature for 1 hour. The mixture wascooled, 20 ml of saturated aqueous NaHCO₃ solution added and the mixturewas concentrated in vacuo. The residue was dissolved in EtOAc and theorganic layer washed with H₂O and brine. The organic layer was driedover anhydrous MgSO4 and evaporated to dryness. To deprotect the acetategroups, the residue was dissolved in 130 ml of MeOH. To this solution, asolution of 230 mg Na in 5 ml MeOH was added. It was stirred at roomtemperature for 1 hour. Dowex 50 (H⁺ form) was added until pH 7. Theresin was filtered and the solution was concentrated to a volume of 50mL.

Crystallization of the product gave 6.2 g of solid title C compound(21.6 mmol), m.p. 184.6-185.5° C.

Elemental analysis calc'd for C₁₂H₁₅N₂O₄Cl: C, 50.40; H, 5.39; N, 9.48;Cl, 12.00. Found: C, 50.78; H, 5.28; N, 9.59; Cl, 12.19.

D.10-[2-[[4-[[(2,3-Dihydroxypropyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

DO3A sulfate (6.0 g, 13.5 mmol) was dissolved in 200 ml of H₂O and thepH of the solution was adjusted to 9.8 by adding 10 N NaOH. To thissolution at 85° C. was added a solution of the title C compound (5.8 g,20.2 mmol) in 200 ml of EtOH over a period of 45 minutes. The pH wasmaintained at 9.8 by adding 5 N NaOH. As the reaction proceeded, themixture turned clear. The mixture was heated at 85° C. for 26 hours. Thesolvents were removed in vacuo. The crude material was dissolved in 500ml of H2O and applied to a 2-liter column of anion exchange resins. Thecolumn was eluted with a gradient of Et₃NH⁺⁻HCO3 buffer, 5 mM to 200 mM.The fractions containing the desired compound were combined andconcentrated in vacuo. The title D compound (6.1 g) was obtained as themono triethylammonium salt (8.8 mmol).

Elemental analysis calc'd for C₃₂H₅₅N₇O₁₀.0.29H₂O: C, 54.67; H, 7.97; N,13.95. Found: C, 54.71; H, 8.14; N, 13.94.

E.10-[2-[[4-[[(2,3-Dihydroxypropyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

700 mg (1.0 mmol) of the title D compound (mono triethylammoniun salt)was dissolved in 10 ml of H₂O and the pH of the solution adjusted to 4.5by adding diluted HOAc. To this solution was added a solution ofGd(OAc)₃.4H₂O (540.4 mg, 1.3 mmol) in 15 ml of H₂O. The mixture wasstirred at 45° C. for 24 hours. The solution was then diluted andapplied to a column of CHP-20 resin. The column was eluted with H₂O,then with increasing amount of EtOH (5-20%). Evaporation of the combinedfractions containing the desired product afforded 300 mg of the puretitle compound (0.40 mmol).

Elemental analysis calc'd for C₂₆H₃₇N₇O₁₀Gd.0.82H₂O: C, 40.79; H, 5.09;N, 10.98. Found: C, 40.81; H, 5.14; N, 10.91.

EXAMPLE 610-[N-(4-Nitrophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt A.10-[N-(4-Nitrophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

A solution of 2-chloro-4′-nitroacetanilide (3 g, 14 mmol) in DMSO (30ml) was slowly added into a solution of DO3A (5.8 g, 16.8 mmol) in water(30 ml) whose pH was adjusted to 10 by the addition of 10 N NaOH at 50°C. The reaction was maintained at 50°-60° C. and the pH was kept at 10for 54 hours. The yellow precipitate was filtered and dissolved in water(150 ml). The pH of the resulting solution was adjusted to ca.2 by theaddition of 1.0 N HCl. The resulting solution was then applied to acolumn of CHP-20P resin. The column was eluted with water (3 L),followed by 5% (1 L). 10% (1 L) and 20% (1.5 L) EtOH containing water inthe order maintained. The fractions containing the desired compound werecombined and concentrated in vacuo to give the yellow title A product(2.6 g).

Analysis calc'd for C₂₂H₃₂N₆O₉ 1.30 H₂O: C, 48.23; H, 6.36; N, 15.34; O,30.07%. Found: C, 47.94; H, 6.48; N. 15.72; H₂O, 4.26%.

B. 10-[N-(4Nitrophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

The title A free acid (580 mg, 1.114 mmol) suspended in water (5 mL) wastreated with gadolinium acetate (602 mg, 1.48 mmol. 1.33 eq.) in water(3.5 mL) at 65° C. Upon mixing the starting materials the solutionbecame homogeneous but after 25 minutes a pale yellow solid precipitatedout; Filtration and washing of the solid with water gave the titleproduct (470 mg).

Analysis calc'd for C₂₂H₂₉N₆O₉Gd.0.69 H₂O: C, 38.23; H, 4.43; N, 12.16;O, 22.41%. Found: C, 38.34; H, 4.48; N. 12.09; H₂O, 1.80%.

EXAMPLE 710-[N-(4-Aminophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt A.10-[N-(4-Aminophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monotriethylammonium salt

To a solution of compound A of Example 6 (5.3 g, 10.1 mmol) in water(150 ml) whose pH was adjusted to 7.0 by the addition of 10 N NaOH wasadded 10% Pd/C catalyst (2.17 g, 1.0 mmol of Pd). The solution washydrogenated at room temperature under a hydrogen atmosphere (20-25 psi)for 3 hours. The reaction mixture was then filtered to remove thecatalyst. The filtrate was concentrated and applied on a 5×20 cm columnof anion exchange resin. The column was eluted with a step gradient (5mM-10 mM) of aqueous triethylammonium bicarbonate solution. Thefractions containing the desired compound were combined and concentratedto yield 4.2 g of the title A mono-triethylammonium salt.

Analysis calc'd for C₂₈H₄₉N₇O₇.1.91 H₂O.0.34 NCC₂H₅l₃ C, 54.29; H, 8.78;N, 15.47; ), 21.45%. Found: C, 53-92; H, 9.18; N, 15-58; H₂O, 5-45%(H-NMR spectrum supports the presence of 0.34 mol-equivalent ofN(C₂H5)₃).

B.10-[N-(4-Aminophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

To a solution of the Compound B of example 6 (3.39 g, 5 mmol) in MeOH(95 ml) and water (18 ml) was added 10% Pd/C catalyst (1.06 g, 0.5 mmolof Pd). The solution was hydrogenated at room temperature under hydrogenatmosphere (20-25 psi) for 10 hours. The solution containing thecatalyst was then filtered. After the filtrate was evaporated todryness, the residue was crystallized from MeOH (30 ml) to give theproduct (3.04 g).

Analysis calc'd for C₂₂H₃₁N₆O₇Gd.4.18 H₂O: C, 36.49; H, 5.48; N, 11.61;O, 24.70%. Found: C, 36.22; H, 5.41; N, 11.41; H₂O, 10.4%.

EXAMPLE 810-[[N-(4-(N′-Isothiocyanto)phenyl]acetamido]]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

This activated species was prepared in situ as follows:

To an aqueous solution (7 ml) of the compound of Example 7.B (194.7 mg,0.3 mmol) was added thiophosgene (138 mg, 1.2 mmol) in CHCl₃ (6 ml). Thebiphasic mixture was stirred at room temperature until the startingmaterial was consumed completely. The aqueous layer was separated, andits pH (1.3) was adjusted to 5.9 by the addition of 1.0 N NaOH. Massspectral analysis of the solution showed the presence of a peak at m/e692 corresponding to Example 8. and

EXAMPLE 910-[N-[4-(N′-Methylthioureido)phenyl]acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

To a solution of the title compound from Example 7 (194.7 mg, 0.3 mmol)in H₂O (7.5 ml) was added a solution of thiophosgene (138 mg, 1.2 mmol)in CHCl₃ (6 ml). The biphasic mixture was stirred at room temperatureuntil the compound was consumed completely. The aqueous layer (pH1.0-1.5) was separated, and the CHCl₃ layer was washed with water (1ml×2). The combined aqueous layers were treated with 1 N NaOH to adjustthe pH of the so-formed title 8 solution to 6.0. Methylamine (18.04 mg,0.58 mmol) was then added, and the reaction mixture was stirred for 10minutes. The resulting solution was loaded on a CHP-20 column and elutedwith water and ethanol. The desired title 9 compound was eluted out by10% of ethanol to give the desired product (129 mg).

Analysis calc'd for C₂₄H₃₄N₇O₇SGd.2.99 H₂O: C, 37.16; H, 5.19; N, 12.64;O, 20.60%. Found: C, 37.00; H, 5.16; N, 12.39; H₂O, 6.94%.

EXAMPLE 1010-[N-[4-(N′,N′-Diethylaminothioureido)phenyl]-acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

To a solution of the title gadolinium chelate of Example 7 (324 mg, 0.5mmol) in H₂O (15 ml) was added a solution of chiophosgene (230 mg, 2mmol) in CHCl₃ (10 ml). The biphasic mixture was stirred at roomtemperature until the chelate was consumed completely to provide asolution of the Example 8 isothiocyanato product. The aqueous layer (pH1.0-1.5) was separated, and the CHCl₃ layer was washed with water (2ml×2). The combined aqueous layers were treated with 1 N NaOH to adjustthe pH of the isothiocyanto solution to 6.0. Diethylamine (73.1 mg, 1.0mmol) was then added, and the reaction mixture was stirred for 10minutes. The resulting solution was loaded on a CHP 20P column andeluted with water and aqueous ethanol. The desired compound was elutedout by 10% ethanol to give the desired product (286 mg).

Analysis calc'd for C₂₇H₄₀N₇O₇SGd 2.31 H₂O: C, 40.26; H, 5.58; N, 12.17;O, 18.49%. Found: C, 40.30; H, 5.71; N, 11.99; H₂O, 5.16%.

EXAMPLE 1110,10′[[[[[(1,2-Ethanediyl)diimino]bis(thioxomethyl)-diimino]bis(4,1-phenylene)]diiminobis[1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid], gadolinium (1:2) salt

The Example 8 isothiocyanato derivative solution was prepared as inExamples 8, 9 and 10. Ethylenediamine (11.2 mg, 0.19 mmol) dissolved inwater (1.0 mL) was added to this solution. The pH of the resultingmixture was initially increased to 10.04 and then decreased to 7.88 atthe end of 3 hours stirring. Concentrated ammonium hydroxide was used toquench the excess Example 8 chelate. The crude product, obtained afterremoval of the water and ammonium hydroxide, was purified by CHP 20Pchromatography. The desired product was eluted out by 10% ethanol togive the dimeric gadolinium chelate (150 mG).

Analysis calc'd for C₄₈H₆₆N₁₄O₁₄S₂Gd₂.2.19 H₂O: c, 38.92; H, 4.79; N,13.24; O, 4.49; S, 4.33%. Found: C, 39.07; H, 4.77; N, 13.19; S, 3.95;H₂O, 2.66%.

EXAMPLE 1210,10′-[[[[(Thioxomethyl)bis(imino)bis(4,1-phenylene)]bis(imino)]bis(2-oxo-2,1-ethanediyl)]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, gadolinium (1:2) salt

The product of Example 7 (194.7 mg, 0.3 mmol) dissolved in water (0.5ml) was added to the Example 8 isothocyanato solution described above.The reaction mixture was stirred at room temperature for 10 hours. Theresulting solution was applied to a CHP 20P column. The column waseluted with H₂O, 2%, 4% and 6% of EtOH containing water in the ordermentioned. The desired compound was eluted by 6% of EtOH to give thetitle product (259 mg).

Analysis calc'd for C₄₅H₆₀N₁₂O₁₄SGd₂.4.37H₂O: C, 38.11; H, 4.88; N,11.85; O, 20.72, S, 2.2b %. Found: C, 38.35; H, 5.03; N, 11.80; H₂O,5.55%.

EXAMPLE 1310,10′,10″-[[[[[[Iminobis(2,1-ethanediyl)triimino]-tris(thioxomethyl)]-triimino]-tris-(4,1-phenylene)]triimino]tris(2-oxo-2,1-ethanediyl)]tris[1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid], gadolinium (1:3) salt

Tris(2-aminoethyl)amine (19.7 mg, 0.135 mmol) dissolved in water (0.5mL) was added to the Example 8 isothiocyanato solution described above.The pH of the resulting mixture read 10.10 upon mixing, and thendecreased to 7.88 after stirring 18 hours. Concentrated ammoniumhydroxide was added to quench the excess Example 8 compound. The crudeproduct, which was obtained after removal of the water and ammoniumhydroxide, was purified by CHP 20P chromatography. The desired productwas eluted by 20% ethanol to give the trimeric gadolinium chelate (230mG).

Analysis calc'd for C₇₅H₁₀₅N₂₂O₂₁S₃Gd₃.6.92H₂O: C, 38.44; H, 5.11; N,13.15; O, 19.0b; S, 4.10%. Found: C, 38.75; H, 5.09; N, 13.14; S, 3.76;H₂O, 5.32%.

EXAMPLE 1410-[2-[[2-(4-Nitrophenyl)ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt A.2-Chloro-N-[2-(4-nitrophenyl)ethyl]acetamide

To a solution of p-nitrophenethylamine (hydrochloride salt, 6.0 g, 29.7mmol) in anhydrous DMA (50 ml) and Et₃N (3.0 g, 29.7 mmol) was addedchloroacetyl chloride (6.71 g, 59.4 mmol). The reaction mixture wasstirred at room temperature for 2 hours. The solvent was removed invacuo, the residue was dissolved in EtOAc and the solution was washedwith aqueous NaHCO₃ and brine. The organic layer was dried and thesolvent removed to obtain the crude product as a yellow solid.Recrystallization of this material from hot EtOAc/hexanes (10:1)afforded the anilide as a white crystal (5.5 g).

Elemental analysis calc'd C₁₀H₁₁N₂ClO₃: C, 49.50; H, 4.57; N, 11.54; Cl,14.61; O, 19.78%. Found: C, 49.74; H, 4.50; N, 11.12; Cl, 14.35%.

B.10-[2-[[2-(4-Nitrophenyl)ethyl]amino]-2-oxoethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

DO3A sulfate (6.0 g, 13.5 mmol) was dissolved in H₂O (100 ml) and the pHof the solution was adjusted to 9.5 by adding 10 N NaOH. To thissolution at 80° C. was added a solution of the title A compound (5.5 g,22.7 mmol) in EtOH (80 ml) over a period of 30 minutes. The pH wasmaintained at 9.5 by adding 5 N NaOH. The mixture was heated at 80° C.for 48 hours, and the solvents were removed in vacuo. The solid wasdissolved in H₂O and washed twice with EtOAc. The aqueous layer wasevaporated by a water pump at 40° C. to remove traces of EtOAc. Thesolution was diluted to 600 ml and applied to a 1.5-liter column ofanion exchange resin. The column was eluted with a step gradient ofaqueous Et₃NH⁺⁻HCO₃ solution, 5 mM to 200 mM. The fractions containingthe desired compound were combined and concentrated in vacuo. The titleB compound (7.67 g) was obtained as the mono triethylammonium salt.

C.10-[2-[[2-(4-Nitrophenyl)ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt

The title B compound (monotriethylammonium salt, 65.3 mg, 0.1 mmol) wasdissolved in H₂O (5 ml) and the pH of the solution adjusted to 4.5 byadding diluted HOAc. To this solution was added a solution ofGd(OAc)₃.4H₂O (52.8 mg, 0.13 mmol) in H₂O (5 ml). The mixture wasstirred at room temperature for 1 hour. The solution was then applied toa 400 ml column of CHP-20 resin, using EtOH/H₂O (0-15%) as an eluent.The fractions containing the desired compound were combined and removalof the solvent afforded the title compound as a monogadolinium salt (450mg).

Elemental analysis calc'd for C₂₄H₃₃N₆GdO₉.1.93 H₂O: C, 38.88; H, 5.01;N, 11.33; Gd, 21.21; O, 23.57%. Found: C, 38.81; H, 5.15; N, 11.40%.

EXAMPLE 1510-[2-[[3,5-bis[[(2-hydroxyethyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid-gadolinium (III) complex A.N,N′-bis(2-hydroxyethyl)-5-nitro-1,3-benzenedicarboxamide

A solution containing dimethyl-5-nitroisophthalate (23.9 g, 100 mmol)and ethanolamine (13.4 g, 220 mmol) in methanol (300 mL) was refluxedfor 48 hours. The solvent was removed by evaporation under reducedpressure. The crude product was purified by crystallization fromEtOAC:MeOH 1 ml v/v) to afford 20.0 g of pureN,N′-bis(2-hydroxyethyl)-5-nitro-1,3-benzenedicarboxamide as whitecrystals. m.p.=151.3-151.4° C., uncorrected

Elemenatal Analysis: Calculated for C₁₂H₁₅O6N3.0.8 H2O: C, 48.25; H,5.12; N, 14.07; O, 32.57%.

B. N,N′-bis(2-acetoxyethyl)-5-amino-1,3-benzenedicarboxamide

A solution containingN,N′-bis(2-hydroxyethyl)-5-amino-1,3-benzenedicarboxamide (6.0 g, 20mmol) and acetic anhydride (11 mL) in pyridine (50 mL) was stirred atroom temperature for 4 hours. Water (1 mL) was added to decompose theexcess anhydride and the solvent was removed in vacuo. The residue wasdissolved in EtOAc (50 mL) and the solution was successively washed withH₂O (50 mL), 10% HCl (50 mL), and saturated NaCl solution (50 mL). Theaqueous layer was reextracted with EtOAc (50 mL) and the organic layerwas combined with the previous extract. The EtOAc layer was dried overMgSO₄, filtered, and evaporated to dryness. The residue (7.3 g, 19 mmol)was dissolved in hot MeOH (100 mL) and hydrogenated using cat. 10% Pd/Cfor 3 hours at 50 psi H₂. The filtrate was evaporated to dryness andafforded 6.4 g of pure title B product.

Elemental Analysis Calculated for: C₁₆H₂₁O₆N₃: C, 54.70; H, 6.02; N,11.96; O, 27.32%. Found: C, 54.97; H, 6.19; N, 12.10; ROI, 0.12; H₂O,0.000%.

C.N,N′-bis(2-acetyloxy)ethyl]-5-[(chloroacetyl)-amino-1,3-benzenedicarboxamide

Chloroacetyl chloride (3 mL) was added dropwise to an ice-cold solutioncontaining N,N′-bis(2-acetoxyethyl)-5-amino-1,3-benzenedicarboxamide(6.4 g, 18 mmol) in anhydrous DMA (5 mL). After one hour, the reactionmixture was neutralized by the addition of saturated aqueous NaHCO₃ andthe solution was concentrated in vacuo to a syrup. The syrup wasdissolved in EtOAc (100 mL) and was successively washed with H₂O (2×50mL) and brine (50 mL). The organic layer was dried over MgSO₄, filteredand evaporated to yield 6.2 g of pure title C product.

Elemental Analysis calculated for C₁₈H₂₂O₇N₃Cl.0.52 H₂O.0.55 CD₃OD: C,48.75; H, 4.85; N, 9.19; Cl, 7.75; O, 24.50%. Found: C,48.35; H, 5.04;N, 9.30; Cl, 8.20; ROI, 0.00; H₂O), 2.16% (desorption KF).

D.5-[chloroacetyl)amino]-N,N′-bis(2-hydroxy-ethyl)-5-amino-1,3-benzenedicarboxamide

A solution ofN,N′-bis(2-(acetyloxy)ethyl]-5-[(chloroacetyl)amino-1,3-benzenedicarboxamide(6.2 g, 14 mmol) in MeOH (20 mL) was treated with NaOMe (600 mg, 10.5mmol). After 2 hours at room temperature, the mixture was neutralizedwith AG-50W-X2 (H+ form) resin. The resin was removed by filtration andthe solution was evaporated to dryness to afford 4.8 g of crudematerial. An analytical sample of the title D product was crystallizedfrom MeOH.

m.p.=178.4-180.6° C., uncorrected.

Elemental Analysis calcd. for C₁₄H₁₈O₅N₃Cl.0.16 H₂O: C, 48.51; H, 5.33;N, 12.12; Cl, 10.23; O, 23.81%. Found: C, 48.73; H, 5.45; N, 12.16; Cl,10.27; ROI, 0.09; H₂O, 0.83 (desorption KF).

E.10-[2-[[3,5-bis[[(2-hydroxyethyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monosodium salt

A suspension of5-[chloroacetyl)amino]-N,N′-bis(2-hydroxyethyl)-1,3-benzenedicarboxamide(1.0 g, 2.9 mmol) in water (20 mL) was added to a basic solution (pH9.8) of DO3A (692 mg, 2.0 mmol) in water (3 mL) maintained at 80° C. Asmall amount of ethanol (5 mL) was added to during the course of theaddition to aid in the solubilization of the chloroanilide. During thecourse of the reaction, the pH was maintained constant by occasionaladdition of small aliquots of 10N NaOH. The progress of the reaction wasmonitored by HPLC. After 20 hours, the reaction was neutralized to pH6.75 with 1N HCl and applied to a CHP-20 column (150 mL). The productwas eluted with water. Fractions containing pure product were combinedand evaporated to dryness to afford 1.1 g of the title E product as awhite glassy material.

m.p.=176° C. (decomp), uncorrected.

Elemental Analysis calc'd for C₂₈H₄₂O₁₁N₇Na.1.97 H₂O: C, 47.30; H, 6.51;N, 13.79; Na, 3.23; O, 29.17. Found: C, 47.34; H, 6.75; N, 13.45; Na,2.91; ROI, not determined; H₂O, 4.98% (desorption KF).

F.10-[2-[[3,5-bis[[(2-hydroxyethyl)amino]-carbonyl]phenyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid, monogadolinium salt(VII)

A solution containing the title E ligand (200 mg, 306 μmol) in water(100 μL) was adjusted to pH 4.69 by addition of HOAc. A solution ofGd(OAc)₃.4 H₂O (187 mg, 461 μmol) in water (50 μL) was added to theligand and the reaction was stirred for 17 hours at 80° C. The crudeproduct was applied directly onto a CHP-20 column (100 mL). Afterwashing the column with water, the pure product was eluted with 5% EtOHin water. Fractions containing the product were pooled and evaporated toyield 160 mg of pure product.

MS (positive FAB, m/z): (M+H)⁺ at 809 (¹⁵⁸ Gd).

Elemental Analysis: calc'd for C₂₈H₃₈O₁₁N₇Gd.2.72 H₂O: C, 39.34; H,5.12; N, 11.4; Gd, 18.39; O, 25.68%. Found: C, 39.41; H, 5.52; N, 11.19;ROI, 17.85; H₂O, 5.74% (desorption KF).

EXAMPLE 1610,10′,10″,10′″,10″″,10′″″-[[[[[[[[(Nitrilo-tri-2,1-ethanediyl)tris(nitrilo)]hexakis-(2,1-ethanediyl)]hexakis(imino)hexakis-(carbonothioyl)]hexakis-(imino)]hexakis-(4,1-phenylene)]hexakis-(imino)]-hexakis-(2-oxo-2,1-ethanediyl)]hexakis[1,4,7,10-tetra-azacyclododecane-1,4,7-triaceticacid], gadolinium (1:6) salt A.Tris[bis(2-p-toluenesulfonylaminoethyl)-aminoethyl]amine

Tris(2-aminoethyl)amine (0.73 g, 4.96 mmol) and N-tosyl aziridine (5.9g, 30.0 mmol) were added in 5% aqueous EtOH (10 ml). The reactionmixture was stirred at room temperature. Over a period of 2 hours awhite precipitate formed, which made stirring difficult. After anotherportion of 5% aqueous EtOH (10 ml) was added, the reaction mixture wasstirred for 21 hours. The solid was filtered and washed with coldethanol to obtain the crude product (6.3 g). The crude product wasrecrystallized twice from hot acetonitrile (120 ml each) to afford thetitle product, tris[bis(2-p-toluenesulfonylaminoethyl)-aminoethyl]amine(3.4 g) in 52% yield.

HPLC: Retention time 7.7 minutes; YMC C₈ basic 120 Å, 4.6×250 mm; 48%acetonitrile in 25 mM HCl; UV at 254 nm; flow rate, 1 ml/min. IR (KBr):3285, 1599, 1325, 1157, 1094 and 552 cm⁻¹. MS(FAB): m/z: 1329.5 (M+H)⁺;904 [M-CH₂N(CH₂CH₂NHTs)₂]; 438 [CH₂CH₂N(CH₂CH₂NHTs)₂]; 424[CH₂N(CH₂CH₂NHTs)₂].

¹H-NMR (DMSO): δ 2.35 (s, 18H, CH₃); 2.31, 2.65 (t, 36H, CH₂); 7.32-7.67(m, 24H, benzene ring).

¹³C-NMR (DMSO): 22.05 (CH₃); 54.38 (CH₂); 127.57, 130.70, 138.75, 143.61(benzene ring). Anal. Calcd. for C₆₀H₈₄N₁₀O₁₂S₆.0.18 H₂O: C, 54.06; H,6.38; N, 10.51, S, 14.43. Found: C, 53.95; H, 6.37; N, 10.55; S, 14.29.H₂O, 0.25% (desorption Karl-Fisher).

B. Tris[bis(2-aminoethyl)aminoethyl]amine

Tris[bis(2-p-toluenesulfonylaminoethyl)-aminoethyl]amine (1 g, 0.75mmol) was treated with concentrated H₂SO₄ (5 ml) under a N₂ atmosphereat 130° C. for 48 hours. After cooling the reaction mixture to 0° C.,diethyl ether (40 ml) was added in small portions maintaining thetemperature under 10° C. The resulting hygroscopic precipitate wasfiltered and dissolved in H₂O (5 ml). The solution was basified to pH 13with 10 N NaOH and extracted with CH₂Cl₂. The CH₂Cl₂ layer was thendried over Na₂SO₄ and concentrated to obtain the title product,tris[bis(2-aminoethyl)aminoethyl]amine (90 mg).

¹H-NMR(D₂O): 2.47-2.68 (t, 36H, CH₂). ¹³C-NMR(D₂O): 37.51 (CH₂NH₂);50.71 (NCH₂CH₂NH₂); 55.91 (NCH₂CH₂N). MS(FAB): m/z: 427 (M+NA)⁺; 405(M+H)⁺; 288 [MCH₂N(CH₂CH₂NH₂)₂]⁺; 276 [MCH₂CH₂N)CH₂CH₂NH₂)₂)₂]⁺.

C.10,10′,10″,10′″,10″″,10′″″-[[[[[[[[(Nitrilo-tri-2,1-ethanediyl)tris(nitrilo)]hexakis-(2,1-ethanediyl)]hexakis(imino)hexakis-(carbonothioyl)]hexakis-(imino)]hexakis-(4,1-phenylene)]hexakis-(imino)]hexakis-(2-oxo-2,1-ethanediyl)]hexakis[1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid], gadolinium (1:6) salt

Tris[bis(2-aminoethyl)aminoethyl]amine (50 mg, 0.124 mmol) dissolved inwater (1.8 ml) was added to Gd(IPA-DO3A) which was prepared as describedin the following paragraph (and is also described as the title compoundof Example 8 above). The reaction mixture was stirred at roomtemperature for 20 hours. The reaction mixture was then concentrated todryness. The residue was dissolved in H₂O (5 ml), and the pH of theresulting solution adjusted to 11.9 by the addition of 1M NaOH. Theresulting aqueous solution was applied to a CHP 20 P column (2.5×20 cm).The column was eluted with water and water containing 2%, 5%, 10%, 20%and 25% EtOH in the sequence indicated. The desired compound was elutedout by 25% of EtOH to afford the title compound C (234 mg) as anoff-white solid in 46% yield.

C₁₅₆H₂₂₂N₄₆O₄₂S₆.6 Gd. 23.49 H₂O MW 3606.14 FW 4972.82.

HPLC: Retention time 7.9 minutes; AMP-303 ODS 200 Å, 4.6×250 mm; 16%acetonitrile in 50 mM tris and 10 mM EDTA (pH 7.0); UV at 254 nm; flowrate, 1 ml/min. IR (KBr): 3437, 1618, 1508, 1385, 1317 and 1084 cm⁻¹.

MS: m/z: 4550.6 (M+H)⁺; 2227.7 (M+2H)⁺². Anal. Calcd. forC₁₅₆H₂₂₂N₄₆O₄₂S₆Gd₆ .23.49 H₂O: C, 37.68; H, 5.45; N, 12.96, S, 3.87.Found: C, 38.02; H, 5.70; N, 12.83; S, 3.53. H₂O, 8.51% (desorptionKarl-Fisher).

In situ preparation of Gd(IPA-DO3A): To an aqueous solution (22 ml) ofmonogadolinium salt of10-[N-(4-aminophenyl)acetamido]-1,4,7,10-tetraazacyclododecane-1,4,7-tri-aceticacid, Gd(APA-DO3A) (603.3 mg, 0.93 mmol) was added a solution ofthiophosgene (427.7 mg, 3.72 mmol) in CHCl₃ (18 ml). The biphasicmixture was stirred at room temperature and the progress of the reactionmonitored by HPLC using a Nucleosil C₁₈ column. When the conversion tothe expected product was completed, the aqueous layer was separated, andits pH (1.0) adjusted to 8 by the addition of 1.0 N NaOH.

Abbreviations used herein:

DMA=dimethylacetamide

THF=tetrahydrofuran

MeI=methyl iodide

EtOAc=ethyl acetate

MeOH methanol

EtOH=ethanol

HOAc=acetic acid

Ac=acetyl

DMSO=dimethylsulfoxide

NaOMe=sodium methoxide

1. A compound of the formula

wherein A₁ is —(CH₂)_(m)′ or a single bond; (CH₂)_(m) and (CH₂)_(m)′ mayindependently be substituted with alkyl or hydroxyalkyl; R₁ and R₂ areeach independently hydrogen or

 with the proviso that at least one of R₁ and R₂ must be other thanhydrogen; R₃ and R₄ are independently hydrogen, alkyl, arylalkyl, aryl,alkoxy and hydroxyalkyl; R₁₂ is hydrogen, alkyl or hydroxyalkyl; R₁₃ ishydrogen, alkyl, arylalkyl, aryl, alkoxy or hydroxyalkyl; m and m′ areindependently 1 to 5; and multimeric forms thereof.
 2. The compound ofclaim 1 having the name10-[2-[[3,5-Bis-[[(2-hydroxy-1-(hydroxymethyl)ethyl]amino]carbonyl]phenylamino]2-oxoethyl]1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid.
 3. The gadolinium complex of the compound of claim
 2. 4. Thecompound of claim 1 having the name10-[2-[[3,5-Bis[[(2-methylbutyl)amino]carbonyl]-phenyl]amino]2-oxoethyl]1,4,7,10-tetraazacyclo-dodecane-1,4,7-triaceticacid.
 5. The gadolinium complex of the compound of claim 4.